Microbial products and uses thereof to improve oil recovery

ABSTRACT

This invention relates to compositions and methods of microbial enhanced oil recovery using biochemical-producing microbes. In specific embodiments, the methods of the subject invention comprise applying a bio surfactant-producing bacteria and/or a growth by-product thereof to an oil-producing site. In preferred embodiments, the bacteria is a strain of Bacillus in spore form. In some embodiments, the methods further comprise applying the bacteria with a yeast fermentation product, an alkaline compound, a polymer, a non-biological surfactant, and/or one or more chelating agents. Advantageously, the subject invention can be useful for stimulating the flow of oil from a well, as well as dissolving scale present in an oil-bearing formation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage Application of InternationalApplication No. PCT/US2018/026724, filed Apr. 9, 2018; which claims thebenefit of U.S. provisional application Ser. No. 62/483,425, filed Apr.9, 2017, both of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

The high demand for fossil fuels necessitates efficient production ofoil. As oil wells mature, it becomes more difficult and costly tocontinue to pump oil at an economically viable rate. Therefore, there isa need to develop improved methods of oil recovery. One such mechanismutilizes microbes and their by-products.

Oil exists in small pores and narrow fissures within the body ofreservoir rocks underneath the surface of the earth. Natural pressure ofthe reservoir causes the oil to flow up to the surface, therebyproviding primary production; however as oil production progresses, thereservoir pressure is depleted to a point at which artificial lift orpumping is required to maintain an economical oil production rate.

When it is necessary to provide external energy for the reservoir toachieve additional oil recovery (secondary recovery), the extra energycan be introduced by injecting gas (gas injection) and/or water (waterflooding). After some years of operation in a field, the injected fluidsflow preferentially along high permeable layers that cause these fluidsto by-pass oil saturated areas in the reservoir. Therefore, anincreasing quantity of water (or gas) rises with the oil and, bydecreasing the ratio of oil to water, eventually it becomes uneconomicalto continue the process; at that time the field must be abandoned.

Primary recovery generally results in an average recovery of only afraction of the oil originally present in an oil bearing formation.Secondary recovery, e.g., water flooding, generally recovers another 10%by the time it becomes uneconomical to continue. It is not unusual for60 to 70% of the oil originally in the formation to remain, even aftersecondary recovery reaches is economical limit. In this situation, athird stage of oil recovery, so-called tertiary production, can beconsidered.

At this tertiary stage, technically advanced methods are employed toeither modify the properties of reservoir fluids or the reservoir rockcharacteristics. In general, the methods can be classified into fourmain categories: thermal methods, chemical methods, miscible or solventinjection, and microbial methods.

Microbial Enhanced Oil Recovery (MEOR) is a multidisciplinary fieldincorporating, inter alia, geology, chemistry, microbiology, fluidmechanics, petroleum engineering, environmental engineering and chemicalengineering. MEOR uses microorganisms and/or their metabolites toenhance the recovery of oil. The microbial methods used in MEOR canaddress well bore clean-up in order to remove mud and other debrisblocking the channels where oil flows; well stimulation improves theflow of oil from the drainage area into the well bore; and enhancedwater floods increase microbial activity by injecting selected microbes,and sometimes nutrients.

In MEOR, nutrients and suitable microbes, which preferably grow underthe anaerobic reservoir conditions, are injected into the reservoir.Microbial by-products, which can include biosurfactants, biopolymers,acids, solvents, gases, and enzymes, for example, can modify theproperties of the oil and the interactions between oil, water, and theporous media, alter the permeability of subterranean formations, andultimately increase the mobility and recovery of oil.

Interest in microbial surfactants has been steadily increasing in recentyears due to their diversity, environmentally friendly nature,possibility of large-scale production, selectivity, performance underextreme conditions, and potential applications in environmentalprotection. Microbially produced surfactants, i.e., biosurfactants,reduce the interfacial tension between water and oil and, therefore, alower hydrostatic pressure is required to move the liquid entrapped inthe pores to overcome the capillary effect. Secondly, biosurfactantscontribute to the formation of micelles providing a physical mechanismto mobilize oil in a moving aqueous phase.

There is a continuing need for improved methods of oil recovery,particular methods that can be sustained for extended periods of time.This includes needs for improved methods of enhanced oil recovery, suchas methods using, for example, microorganisms and/or their growthby-products. Biosurfactants enhance the emulsification of hydrocarbons,have the potential to solubilize hydrocarbon contaminants and increasetheir availability for microbial degradation. These compounds can alsobe used in enhanced oil recovery.

BRIEF SUMMARY OF THE INVENTION

In certain embodiments, the subject invention provides microbes, as wellas substances, such as biosurfactants, solvents and/or enzymes, derivedfrom these microbes and the fermentation broth in which they areproduced. The subject invention also provides methods of using thesemicrobes and their by-products in improved oil production.

Specifically, the subject invention provides cost-effective,environmentally-friendly approaches to enhancing oil recovery.Advantageously, these methods can be practiced over a wide range oftemperatures, including from 20 to 70° C., and higher.

In some embodiments, the subject invention provides materials andmethods for improving oil production by treating an oil-producing site,e.g., an oil-bearing formation or an oil well, with microorganismsand/or their growth by-products. In one embodiment, the subjectinvention can be useful for enhancing oil recovery from an oil well by,e.g., stimulating the flow of oil from the well while dissolving scalewithin the formation.

In some embodiments, the present invention utilizes yeast growthby-products, such as, for example, biosurfactants. Biosurfactants areuseful in the oil and gas industry for their ability to enhance oilrecovery. Biosurfactants can modify the properties of the oil and theinteractions between oil, water, and the porous media in which oil andgas originate, thereby increasing the mobility, and consequently therecovery, of oil. Thus, the compositions and methods of the subjectinvention can increase recovery of crude oil and natural gas from oiland gas containing formations by dramatically reducing both the surfaceand interfacial tension between substances within the formations and byaltering the wettability of formations.

In one embodiment, the subject invention provides yeast fermentationproducts for enhancing oil recovery from an oil-bearing formation. Inone embodiment, the yeast fermentation product is obtained throughcultivation of biosurfactant-producing yeast using processes rangingfrom small to large scale. The cultivation process can be, for example,submerged cultivation, solid state fermentation (SSF), and/or acombination thereof. In one embodiment, yeast products are cultivatedusing a simplified yeast fermentation technique, which reducescultivation time by 50% and reduces carbon source supplementation.

The yeast fermentation product can be obtained via cultivation of abiochemical-producing yeast, such as, for example, Pichia anomala(Wickerhamomyces anomalus). The fermentation broth after 7 days ofcultivation at 25-30° C. can contain the yeast cell suspension and, forexample, 4 g/L or more of glycolipid biosurfactants.

The yeast fermentation product can also be obtained via cultivation ofthe biosurfactant-producing yeast, Starmerella bombicola. Thefermentation broth after 5 days of cultivation at 25° C. can contain theyeast cell suspension and, for example, 150 g/L or more of glycolipidbiosurfactants.

The yeast fermentation product can comprise the fermentation broth,separated from the yeast cells. In one embodiment, the biosurfactants orother growth by-products in the broth are further separated from thebroth and purified.

In some embodiments, the subject invention utilizes strains of bacteriaand by-products thereof. These by-products can include, for example,metabolites, polymers, biosurfactants, enzymes, organic acids, andsolvents. In certain embodiments, the bacteria are strains of Bacillusthat thrive in high salt environments, such as those often encounteredat an oil extraction site. In certain embodiments, the bacteria aresurfactant over-producing strains of Bacillus, meaning such strains arecharacterized by enhanced biosurfactant production compared to wild typeBacillus strains. In certain embodiments, the Bacillus strains haveincreased enzyme production.

In one embodiment, the microorganisms are strains of Bacillus subtilis,Bacillus licheniformis, and/or Bacillus amyloliquefaciens. In preferredembodiments, the bacteria are in spore form.

In some embodiments, the Bacillus strains are capable of thriving underlow oxygen conditions, thereby facilitating growth under microaerophilicand anaerobic conditions. Under anaerobic conditions, nitrate salts canbe added to replace oxygen as an electron acceptor to supportmicroaerophilic and/or anaerobic respiration.

In one embodiment, the Bacillus subtilis strains are, for example, B.subtilis var. locuses strains B1 and B2, which are effective producersof the amphiphilic lipopeptide surfactin.

In one embodiment the subject invention provides a method for improvingoil recovery by applying one or more microorganisms capable of producinguseful biochemical byproducts to an oil-producing site, e.g., anoil-bearing formation and/or oil well. The method optionally includesadding nutrients and/or other agents to the site. In certainembodiments, the microorganisms are selected from strains of Bacillus,including, but not limited to, strains of Bacillus subtilis, Bacilluslicheniformis, and Bacillus amyloliquefaciens. In preferred embodiments,the bacteria are in spore form.

The method may also comprise adding a yeast fermentation product, suchas the fermentation broth resulting from cultivation of, e.g.,Starmerella bombicola or Wickerhamomyces anomalus. In one embodiment,the yeast cells can be removed from the yeast fermentation product andonly the broth containing biosurfactants and other cell exudates isapplied. In one embodiment, the yeast fermentation product comprisesbiosurfactants that have been separated from the fermentation broth andpurified.

The method may also comprise applying the microbes and/or microbialgrowth by-products with one or more alkaline compounds. The alkalinecompound can be, for example, ammonium hydroxide.

In some embodiments, the method may also comprise applying the microbesand/or microbial growth by-products with one or more polymer compounds.The polymer compounds can be selected from biopolymers such as, forexample, hydrogels, polysaccharides, xanthan gum, guar gum, andcellulose polymers.

In some embodiments, the method may also comprise applying the microbesand/or microbial growth by-products with one or more non-biologicalsurfactants. The surfactants may be, for example, anionic, cationic,non-ionic or zwitterionic.

In one embodiment, the microbes and/or microbial growth by-products canbe applied with one or more chelating agents for reducing, e.g.,dissolving, scale that has accumulated within an oil-bearing formation.The chelating agents may be, for example, citric acid, EDTA, sodiumcitrate and/or a combination thereof.

In one embodiment, the microorganisms can germinate and grow in situwithin an oil-bearing formation or oil well, and produce biosurfactantstherein. Consequently, a high concentration of biosurfactants andbiosurfactant-producing microorganisms at a treatment site (e.g., an oilwell) can be achieved easily and continuously.

In some embodiments, the subject microbe-based products and methods canfurther be used for paraffin removal, liquefaction of solid asphaltene,and bioremediation of hydrocarbon-contaminated waters, soils and othersites. For such uses, the methods can further comprise adding solvents,such as isopropyl alcohol or ethanol, with the microbes and/or microbialgrowth by-products.

In one embodiment, the subject invention provides methods of producing abiosurfactant by cultivating a microbe strain of the subject inventionunder conditions appropriate for growth and biosurfactant production;and purifying the biosurfactant. The subject invention also providesmethods of producing solvents, enzymes or other proteins by cultivatinga microbe strain of the subject invention under conditions appropriatefor growth and solvent, enzyme or protein expression; and purifying thesolvent, enzyme or other protein.

The microbe-based products of the subject invention can be used in avariety of unique settings because of, for example, the ability toefficiently deliver fresh fermentation broth with active metabolites; amixture of cells and fermentation broth; compositions with a highdensity of cells; microbe-based products on short-order; andmicrobe-based products in remote locations.

Advantageously, the present invention can be used without releasinglarge quantities of inorganic compounds into the environment.Additionally, the claimed compositions and methods utilize componentsthat are biodegradable and toxicologically safe. Thus, the presentinvention can be used in oil and gas production (and other industries)as an environmentally-friendly treatment.

DETAILED DESCRIPTION

In certain embodiments, the subject invention provides microbes, as wellas substances, such as biosurfactants, solvents and/or enzymes, derivedfrom these microbes and the fermentation broth in which they areproduced. The subject invention also provides methods of using thesemicrobes and their by-products in improved oil production.

Specifically, the subject invention provides cost-effective,environmentally-friendly approaches to enhancing oil recovery.Advantageously, these methods can be practiced over a wide range oftemperatures, including from 20 to 70° C., and higher.

In some embodiments, the subject invention provides materials andmethods for improving oil production by treating an oil-producing sitewith microorganisms and/or their growth by-products. In one embodiment,the subject invention can be useful for enhancing oil recovery from anoil-bearing formation or an oil well by, e.g., stimulating the flow ofoil from the formation or well while dissolving scale therein.

In some embodiments, the present invention utilizes yeast growthby-products, such as, for example, biosurfactants. Biosurfactants areuseful in the oil and gas industry for their ability to enhance oilrecovery. Biosurfactants can modify the properties of the oil and theinteractions between oil, water, and the porous media in which oil andgas originate, thereby increasing the mobility, and consequently therecovery, of oil. Thus, the compositions and methods of the subjectinvention can increase recovery of crude oil and natural gas from oiland gas containing formations by dramatically reducing both the surfaceand interfacial tension between substances within the formations and byaltering the wettability of formations.

In specific embodiments, the methods and compositions described hereinutilize microorganisms to enhance recovery of oil. The microorganismscan improve the quantity and quality of oil recovered from oilreservoirs, including those that are considered “mature.” Furthermore,the microorganisms can remove toxic substances from oil productionsites.

In some embodiments, the subject invention provides materials andmethods for improving oil production by treating an oil-producing sitewith microorganisms and/or their growth by-products. In one embodiment,the subject invention can be useful for enhancing oil recovery from anoil-bearing formation or an oil well by, e.g., stimulating the flow ofoil from the well or formation.

In one embodiment the subject invention provides a method for improvingoil recovery by applying one or more microorganisms capable of producinguseful biochemical byproducts to an oil-producing site, e.g., anoil-bearing formation and/or oil well. The method optionally includesadding nutrients and/or other agents to the site. In certainembodiments, the microorganisms are selected from strains of Bacillus,including, but not limited to, strains of Bacillus subtilis, Bacilluslicheniformis, and Bacillus amyloliquefaciens. In preferred embodiments,the bacteria are in spore form.

In certain embodiments, the bacteria are strains of Bacillus that thrivein high salt environments, such as those often encountered at an oilextraction site. In certain embodiments, the bacteria are surfactantover-producing strains of Bacillus, meaning such strains arecharacterized by enhanced biosurfactant production compared to wild typeBacillus strains. In certain embodiments, the Bacillus strains haveincreased enzyme production.

The method may also comprise adding yeast fermentation products, such asthe fermentation broth resulting from cultivation ofbiochemical-producing yeasts, e.g., Starmerella bombicola orWickerhamomyces anomalus. In one embodiment, the yeast fermentationproduct comprises purified biosurfactants produced by these yeasts.

The method may also comprise applying the microbes with one or morealkaline compounds, polymers, surfactants and/or chelating agents.

Selected Definitions

As used herein, reference to a “microbe-based composition” means acomposition that comprises components that were produced as the resultof the growth of microorganisms or other cell cultures. Thus, themicrobe-based composition may comprise the microbes themselves and/orby-products of microbial growth. The microbes may be in a vegetativestate, in spore form, in mycelial form, in any other form of propagule,or a mixture of these. The microbes may be planktonic or in a biofilmform, or a mixture of both. The by-products of growth may be, forexample, metabolites (e.g., biosurfactants), cell membrane components,expressed proteins, and/or other cellular components. The microbes maybe intact or lysed. The cells may be absent, or the cells may be presentat, for example, a concentration of 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, or 1×10¹¹ or more cells or propagules per milliliter ofthe composition. As used herein, a propagule is any portion of amicroorganism from which a new and/or mature organism can develop,including but not limited to, cells, conidia, cysts, spores (e.g.,reproductive spores, endospores and exospores), mycelia, buds and seeds.

The subject invention further provides “microbe-based products,” whichare products that are to be applied in practice to achieve a desiredresult. The microbe-based product can be simply the microbe-basedcomposition harvested from the microbe cultivation process.Alternatively, the microbe-based product may comprise furtheringredients that have been added. These additional ingredients caninclude, for example, stabilizers, buffers, appropriate carriers, suchas water, salt solutions, or any other appropriate carrier, addednutrients to support further microbial growth, non-nutrient growthenhancers, such as plant hormones, and/or agents that facilitatetracking of the microbes and/or the composition in the environment towhich it is applied. The microbe-based product may also comprisemixtures of microbe-based compositions. The microbe-based product mayalso comprise one or more components of a microbe-based composition thathave been processed in some way such as, but not limited to, filtering,centrifugation, lysing, drying, purification and the like.

As used herein, “harvested” refers to removing some or all of themicrobe-based composition from a growth vessel.

In some embodiments, the microbes used according to the subjectinvention are “surfactant over-producing.” For example, the strain mayproduce at least 0.1-10 g/L, e.g., 0.5-1 g/L surfactant. For example,the bacteria produce at least 10%, 25%, 50%, 100%, 2-fold, 5-fold, 7.5fold, 10-fold, 12-fold, 15-fold or more compared to other oil-recoverymicrobial strains. Specifically, Bacillus subtilis ATCC 39307 is usedherein as a reference strain.

As used herein, an “isolated” or “purified” nucleic acid molecule,polynucleotide, polypeptide, protein or organic compound such as a smallmolecule (e.g., those described below), is substantially free of othercompounds, such as cellular material, with which it is associated innature. Reference to “isolated” in the context of a strain ofmicroorganism means that the strain is removed from the environment inwhich it exists in nature. Thus, the isolated strain may exist as, forexample, a biologically pure culture, or as spores (or other forms ofthe strain) in association with a carrier. A purified or isolatedpolynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA))is free of the genes or sequences that flank it in itsnaturally-occurring state. A purified or isolated polypeptide is free ofthe amino acids or sequences that flank it in its naturally-occurringstate.

In certain embodiments, purified compounds are at least 60% by weight(dry weight) the compound of interest. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight the compound of interest. For example, a purifiedcompound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%,or 100% (w/w) of the desired compound by weight. Purity is measured byany appropriate standard method, for example, by column chromatography,thin layer chromatography, or high-performance liquid chromatography(HPLC) analysis.

A “metabolite” refers to any substance produced by metabolism or asubstance necessary for taking part in a particular metabolic process. Ametabolite can be an organic compound that is a starting material (e.g.,glucose), an intermediate (e.g., acetyl-CoA) in, or an end product(e.g., n-butanol) of metabolism. Examples of metabolites can include,but are not limited to, enzymes, toxins, acids, solvents, alcohols,proteins, carbohydrates, vitamins, minerals, microelements, amino acids,polymers, and surfactants.

By “modulate” is meant alter (increase or decrease). Such alterationsare detected by standard art known methods such as those describedherein.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 aswell as all intervening decimal values between the aforementionedintegers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,and 1.9. With respect to sub-ranges, “nested sub-ranges” that extendfrom either end point of the range are specifically contemplated. Forexample, a nested sub-range of an exemplary range of 1 to 50 maycomprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

By “reduces” is meant a negative alteration of at least 1%, 5%, 10%,25%, 50%, 75%, or 100%.

By “reference” is meant a standard or control condition.

By “salt-tolerant” is meant a microbial strain capable of growing in asodium chloride concentration of fifteen (15) percent or greater. In aspecific embodiment, “salt-tolerant” refers to the ability to grow in150 g/L or more of NaCl.

As used herein, a “biofilm” is a complex aggregate of microorganisms,such as bacteria, wherein the cells adhere to each other. The cells inbiofilms are physiologically distinct from planktonic cells of the sameorganism, which are single cells that can float or swim in liquidmedium.

As used herein, “surfactant” refers to a compound that lowers thesurface tension (or interfacial tension) between two liquids or betweena liquid and a solid. Surfactants act as detergents, wetting agents,emulsifiers, foaming agents, and/or dispersants. A surfactant producedby microorganisms is referred to as a “biosurfactant.”

As used herein, “oil production” refers to any and all operationsinvolved in the extraction of hydrocarbons such as crude oil or naturalgas from a formation through its eventual processing and use byconsumers. Oil production can include, but is not limited to, drilling,pumping, recovery, transmission, processing, refining, transportation,and storage of hydrocarbons.

An “oil-producing site” refers to any environment or structure, whethernaturally-occurring or man-made, wherein one or more aspects ofhydrocarbon, oil and/or natural gas recovery occurs, including but notlimited to, subterranean formations, oil and gas containing formations,wells and wellbores.

As used herein, “scale” refers to accumulations formed by, for example,deposits of precipitated mineral salts, which can arise as a result of,for example, changes in the pressure, composition and/or temperature ofcrude oil. Scales can result from precipitates of, for example, bariumsulfate, calcium carbonate, strontium sulfate, calcium sulfate, sodiumchloride, silicon dioxide, iron sulfide, iron oxides, iron carbonate,silicates, phosphates and oxides, or any of a number of compounds thatare insoluble or mildly soluble in water.

As used herein, “improving oil recovery” includes enhancing recovery ofoil and hydrocarbons, and means increasing the amount of hydrocarbonsproduced and/or increasing the rate at which they are produced, e.g., bystimulating the flow of oil from the well.

The transitional term “comprising,” which is synonymous with“including,” or “containing,” is inclusive or open-ended and does notexclude additional, unrecited elements or method steps. By contrast, thetransitional phrase “consisting of” excludes any element, step, oringredient not specified in the claim. The transitional phrase“consisting essentially of” limits the scope of a claim to the specifiedmaterials or steps “and those that do not materially affect the basicand novel characteristic(s)” of the claimed invention.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a,” “an” and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. All references cited herein are hereby incorporated byreference.

Microbial Growth By-Products According to the Subject Invention

In preferred embodiments, a composition is provided for improving and/orenhancing oil recovery from an oil production site, wherein thecomposition comprises one or more microorganisms and/or growthby-products thereof. In specific embodiments, the microorganisms arecapable of, and are used for, producing one or more biosurfactants.

Biosurfactants are a structurally diverse group of surface-activesubstances produced by microorganisms. Biosurfactants are biodegradableand can be easily and cheaply produced using selected organisms onrenewable substrates. Most biosurfactant-producing organisms producebiosurfactants in response to the presence of hydrocarbon source (e.g.oils, sugar, glycerol, etc) in the growing media. Other media componentssuch as concentration of iron can also affect biosurfactant productionsignificantly.

All biosurfactants are amphiphiles. They consist of two parts: a polar(hydrophilic) moiety and non-polar (hydrophobic) group. Due to theiramphiphilic structure, biosurfactants increase the surface area ofhydrophobic water-insoluble substances, increase the waterbioavailability of such substances, and change the properties ofbacterial cell surfaces.

Biosurfactants accumulate at interfaces, thus reducing interfacialtension and leading to the formation of aggregated micellular structuresin solution. The ability of biosurfactants to form pores and destabilizebiological membranes permits their use as antibacterial, antifungal, andhemolytic agents. Combined with the characteristics of low toxicity andbiodegradability, biosurfactants are advantageous for use in the oil andgas industry for a variety of applications. These applications include,but are not limited to, enhancement of crude oil recovery; reduction ofoil viscosity; paraffin removal from rods, tubing, liners, and pumps;petroleum equipment corrosion prevention; fracturing fluids; reductionof H₂S concentration in extracted crude oil; as well as tank, flowlineand pipeline cleaning.

Safe, effective microbial bio-surfactants reduce the surface andinterfacial tensions between the molecules of liquids, solids, andgases. As discussed herein, this activity can be highly advantageous inthe context of oil recovery.

Biosurfactants produced according to the subject invention can be usedfor other, non-oil recovery purposes including, for example, cleaningpipes, reactors, and other machinery or surfaces.

Biosurfactants include low molecular weight glycolipids (GLs),lipopeptides (LPs), flavolipids (FLs), phospholipids, and high molecularweight polymers such as lipoproteins, lipopolysaccharide-proteincomplexes, and polysaccharide-protein-fatty acid complexes. Thehydrocarbon chain of a fatty acid acts as the common lipophilic moietyof a biosurfactant molecule, whereas the hydrophilic part is formed byester or alcohol groups of neutral lipids, by the carboxylate group offatty acids or amino acids (or peptides), organic acid in the case offlavolipids, or, in the case of glycolipids, by the carbohydrate.

In one embodiment, the microbial biosurfactants according to the subjectinvention include glycolipids such as rhamnolipids (RLP), sophorolipids(SLP), trehalose lipids or mannosylerythritol lipids (MEL).

In one embodiment, the microbial biosurfactant is a lipopeptide, suchas, for example, surfactin or iturin A.

Microbial biosurfactants are produced by a variety of microorganismssuch as bacteria, fungi, and yeasts. Exemplary biosurfactant-producingmicroorganisms include Pseudomonas species (P. aeruginosa, P. putida, P.florescens, P. fragi, P. syringae); Pseudozyma (P. aphidis)Flavobacterium spp.; Pichia spp. (P. anomala, P. lynferdii, P.guilliermondii, P. sydowiorum), Bacillus spp. (B. subtilis, B.amyloliquefaciens, B. pumillus, B. cereus, B. licheniformis);Wickerhamomyces spp. (W. anomalus), Starmerella spp. (S. bombicola),Candida spp. (C. albicans, C. rugosa, C. tropicalis, C. lipolytica, C.torulopsis); Rhodococcus spp.; Arthrobacter spp.; Campylobacter spp.;Cornybacterium spp. and so on. The biosurfactants may be obtained byfermentation processes known in the art.

Growth of Microbes According to the Subject Invention

The subject invention utilizes methods for cultivation of microorganismsand production of microbial metabolites and/or other by-products ofmicrobial growth. The subject invention further utilizes cultivationprocesses that are suitable for cultivation of microorganisms andproduction of microbial metabolites on a desired scale. The microbialcultivation systems would typically use submerged culture fermentation;however, surface culture and hybrid systems can also be used. As usedherein “fermentation” refers to growth of cells under controlledconditions. The growth could be aerobic or anaerobic.

In one embodiment, the subject invention provides materials and methodsfor the production of biomass (e.g. viable cellular material),extracellular metabolites (e.g. small molecules and excreted proteins),residual nutrients and/or intracellular components (e.g. enzymes andother proteins).

The microbe growth vessel used according to the subject invention can beany fermenter or cultivation reactor for industrial use. In oneembodiment, the vessel may have functional controls/sensors or may beconnected to functional controls/sensors to measure important factors inthe cultivation process, such as pH, oxygen, pressure, temperature,agitator shaft power, humidity, viscosity and/or microbial densityand/or metabolite concentration.

In a further embodiment, the vessel may also be able to monitor thegrowth of microorganisms inside the vessel (e.g. measurement of cellnumber and growth phases). Alternatively, a daily sample may be takenfrom the vessel and subjected to enumeration by techniques known in theart, such as dilution plating technique. Dilution plating is a simpletechnique used to estimate the number of bacteria in a sample. Thetechnique can also provide an index by which different environments ortreatments can be compared.

In one embodiment, the method includes supplementing the cultivationwith a nitrogen source. The nitrogen source can be, for example,potassium nitrate, ammonium nitrate ammonium sulfate, ammoniumphosphate, ammonia, urea, and/or ammonium chloride. These nitrogensources may be used independently or in a combination of two or more.

The method of cultivation can provide oxygenation to the growingculture. One embodiment utilizes slow motion of air to remove low-oxygencontaining air and introduce oxygenated air. The oxygenated air may beambient air supplemented daily through mechanisms including impellersfor mechanical agitation of the liquid, and air spargers for supplyingbubbles of gas to the liquid for dissolution of oxygen into the liquid.

The method can further comprise supplementing the cultivation with acarbon source. The carbon source is typically a carbohydrate, such asglucose, sucrose, lactose, fructose, trehalose, mannose, mannitol,and/or maltose; organic acids such as acetic acid, fumaric acid, citricacid, propionic acid, malic acid, malonic acid, and/or pyruvic acid;alcohols such as ethanol, propanol, butanol, pentanol, hexanol,isobutanol, and/or glycerol; fats and oils such as soybean oil, ricebran oil, olive oil, canola oil, coconut oil, corn oil, sesame oil,and/or linseed oil. These carbon sources may be used independently or ina combination of two or more.

In one embodiment, growth factors and trace nutrients for microorganismsare included in the medium. This is particularly preferred when growingmicrobes that are incapable of producing all of the vitamins theyrequire. Inorganic nutrients, including trace elements such as iron,zinc, copper, manganese, molybdenum and/or cobalt may also be includedin the medium. Furthermore, sources of vitamins, essential amino acids,and microelements can be included, for example, in the form of flours ormeals, such as corn flour, or in the form of extracts, such as yeastextract, potato extract, beef extract, soybean extract, banana peelextract, and the like, or in purified forms. Amino acids such as, forexample, those useful for biosynthesis of proteins, can also beincluded, e.g., L-Alanine.

In one embodiment, inorganic salts may also be included. Usableinorganic salts can be potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate,magnesium chloride, iron sulfate, iron chloride, manganese sulfate,manganese chloride, zinc sulfate, lead chloride, copper sulfate, calciumchloride, calcium carbonate, and/or sodium carbonate. These inorganicsalts may be used independently or in a combination of two or more.

In some embodiments, the method for cultivation may further compriseadding additional acids and/or antimicrobials in the liquid mediumbefore, and/or during the cultivation process. Antimicrobial agents orantibiotics can be used for protecting the culture againstcontamination. Additionally, antifoaming agents may also be added toprevent the formation and/or accumulation of foam when gas is producedduring cultivation.

The pH of the mixture should be suitable for the microorganism ofinterest. Buffers, and pH regulators, such as carbonates and phosphates,may be used to stabilize pH near a preferred value. When metal ions arepresent in high concentrations, use of a chelating agent in the liquidmedium may be necessary.

The method and equipment for cultivation of microorganisms andproduction of the microbial by-products can be performed in a batch,quasi-continuous, or continuous processes.

The microbes can be grown in planktonic form or as biofilm. In the caseof biofilm, the vessel may have within it a substrate upon which themicrobes can be grown in a biofilm state. The system may also have, forexample, the capacity to apply stimuli (such as shear stress) thatencourages and/or improves the biofilm growth characteristics.

In one embodiment, the method for cultivation of microorganisms iscarried out at about 5° to about 100° C., preferably, 15 to 60° C., morepreferably, 25 to 50° C. In a further embodiment, the cultivation may becarried out continuously at a constant temperature. In anotherembodiment, the cultivation may be subject to changing temperatures.

In one embodiment, the equipment used in the method and cultivationprocess is sterile. The cultivation equipment such as the reactor/vesselmay be separated from, but connected to, a sterilizing unit, e.g., anautoclave. The cultivation equipment may also have a sterilizing unitthat sterilizes in situ before starting the inoculation. Air can besterilized by methods know in the art. For example, the ambient air canpass through at least one filter before being introduced into thevessel. In other embodiments, the medium may be pasteurized or,optionally, no heat at all added, where the use of low water activityand low pH may be exploited to control bacterial growth.

The biomass content of the fermentation broth may be, for example from 5g/l to 180 g/l or more. In one embodiment, the solids content of thebroth is from 10 g/l to 150 g/l.

In one embodiment, the subject invention further provides a method forproducing microbial metabolites such as ethanol, lactic acid,beta-glucan, proteins, peptides, metabolic intermediates,polyunsaturated fatty acid, and lipids. The metabolite content producedby the method can be, for example, at least 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90%.

The microbial growth by-product produced by microorganisms of interestmay be retained in the microorganisms or secreted into the liquidmedium. In another embodiment, the method for producing microbial growthby product may further comprise steps of concentrating and purifying themicrobial growth by-product of interest. In a further embodiment, theliquid medium may contain compounds that stabilize the activity ofmicrobial growth by-product.

In one embodiment, all of the microbial cultivation composition isremoved upon the completion of the cultivation (e.g., upon, for example,achieving a desired cell density, or density of a specified metabolitein the broth). In this batch procedure, an entirely new batch isinitiated upon harvesting of the first batch.

In another embodiment, only a portion of the fermentation product isremoved at any one time. In this embodiment, biomass with viable cellsremains in the vessel as an inoculant for a new cultivation batch. Thecomposition that is removed can be a cell-free broth or contain cells.In this manner, a quasi-continuous system is created.

Advantageously, the method does not require complicated equipment orhigh energy consumption. The microorganisms of interest can becultivated at small or large scale on site and utilized, even beingstill-mixed with their media. Similarly, the microbial metabolites canalso be produced at large quantities at the site of need.

Advantageously, the microbe-based products can be produced in remotelocations. The microbe growth facilities may operate off the grid byutilizing, for example, solar, wind and/or hydroelectric power.

The microorganisms useful according to the systems and methods of thesubject invention can be, for example, bacteria, yeast and/or fungi.These microorganisms may be natural, or genetically modifiedmicroorganisms. For example, the microorganisms may be transformed withspecific genes to exhibit specific characteristics. The microorganismsmay also be mutants of a desired strain. As used herein, “mutant” meansa strain, genetic variant or subtype of a reference microorganism,wherein the mutant has one or more genetic variations (e.g., a pointmutation, missense mutation, nonsense mutation, deletion, duplication,frameshift mutation or repeat expansion) as compared to the referencemicroorganism. Procedures for making mutants are well known in themicrobiological art. For example, UV mutagenesis and nitrosoguanidineare used extensively toward this end.

In one embodiment, the microorganisms are bacteria, includingGram-positive and Gram-negative bacteria. The bacteria can be endosporeor exospore forming bacteria. The bacteria may be, for exampleAgrobacterium radiobacter, Alcanivora borkumensis, Azobacter (A.vinelandii, A. chroococcum), Azospirillum brasiliensis, Bacillus (e.g.,B. subtilis, B. lichcniformis, B. firmus, B. laterosporus, B.megaterium, B. amyloliquifaciens), Clostridium (C. butyricum, C.tyrobutyricum, C. acetobutyricum, Clostridium NIPER 7, and C.beijerinckii), Lactobacillus fermentum, Norcardia sp., Pseudomonas (P.chlororaphis subsp. aureofaciens (Kluyver), P. aeruginosa), Rhizobium,Rhodospirillum rubrum. Sphingomonas paucimobilis, Ralstonia eulropha,Serratia marcescens and/or Tsukamurella sp.

In preferred embodiments, the microorganism is a strain of Bacillusselected from the species B. subtilis, B. amyloliquefaciens and B.licheniformis. Even more preferably, the strain of Bacillus is in sporeform.

In certain embodiments, the present invention utilizes Bacillus subtilisstrains with enhanced biosurfactant production compared to wild typeBacillus subtilis as well as compared to other microbes used in oilrecovery. In certain embodiments, the Bacillus subtilis strains haveincreased biopolymer, solvent and/or enzyme production. Such Bacillussubtilis have been termed members of the B series, including, but notlimited to, B1, B2 and B3.

In one embodiment, the microorganism is B. subtilis var. locuses B1 orB2, which are effective producers of, for example, surfactin and otherbiosurfactants, as well as biopolymers. This specification incorporatesby reference International Publication No. WO 2017/044953 A1 to theextent it is consistent with the teachings disclosed herein.

A culture of the B. subtilis B1 microbe has been deposited with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.,Manassas, Va. 20110-2209 USA. The deposit has been assigned accessionnumber ATCC No. PTA-123459 by the depository and was deposited on Aug.30, 2016.

The vegetative cells of Bacillus subtilis strain B1 are rods that are0.7 to 0.9 μm wide by 1.6 to 3.3 μm long and occur singly. It is motile,Gram positive and produces biopolymers on nutrient agar and potatodextrose agar. It also produces ellipsoidal spores centrally orparacentrally in unswollen sporangia. The size of mature spores is 0.8to 1.0 μm wide by 1.6 to 1.9 μm long. Agar colonies are cream/beige incolor, raised, mucous, circular, entire, smooth, shiny and 3.0 to 7.0 mmin diameter after 16 hours at 40° C. on nutrient agar plate. It is afacultative aerobe with a growth temperature range of 25-55° C., withoptimal growth temperature at 35° C. It hydrolyzes starch, is positiveon Voges-Proskauer test, can utilize citrate and can grow with 15% NaCl.

In certain embodiments, the Bacillus subtilis strains are salt tolerant.Salt tolerance can be with respect to any one or more of a variety ofsalts. For example, the salt can be a monovalent salt such as a sodiumor potassium salt, e.g., NaCl or KCl, or a divalent salt such as amagnesium or calcium salt, e.g., MgCl₂ or CaCl₂, or a trivalent salt.Given geographic sites to be treated, zinc, bromium, iron, or lithiumsalts are present in the composition or site. In preferred embodiments,the bacteria described herein are tolerant to NaCl as well as others ofthe aforementioned salts and are, therefore, widely useful for oilrecovery.

In some embodiments, the Bacillus subtilis strains are capable ofthriving under low oxygen conditions. In some embodiments, the Bacillussubtilis strain is grown under microaerophilic or anaerobic conditions.Under microaerophilic and/or anaerobic conditions, nitrate salts can beadded to replace oxygen as the electron acceptor to support theanaerobic respiration.

The B strain series of Bacillus subtilis produce more biosurfactantcompared to reference strains of Bacillus subtilis. Furthermore, theBacillus subtilis strains survive under high salt and anaerobicconditions better than other well-known strains. The strains are alsocapable of growing under anaerobic conditions. The Bacillus subtilis Bseries strains can also be used for producing enzymes that degrade ormetabolize oil or other petroleum products.

In one embodiment, the subject methods can utilize products of thefermentation of yeasts or fungi. Yeast and fungus species suitable foruse according to the current invention, include, for example, Candida,Saccharomyces (S. cerevisiae, S. boulardii sequela, S. torula),Issatchenkia, Kluyveromyces, Pichia, Wickerhamomyces (e.g., W.anomalus), Starmerella (e.g., S. bombicola), Rhodotorula (e.g., R.glutinous and R. graminus), Mycorrhiza, Mortierella, Phycomyces,Blakeslea, Thraustochytrium, Phythium, Entomophthora, Aureobasidiumpullulans, Pseudozyma aphidis, Aspergillus, and/or Rhizopus spp.

In one embodiment, the yeast is a killer yeast. As used herein, “killeryeast” means a strain of yeast characterized by its secretion of toxicproteins or glycoproteins, to which the strain itself is immune. Theexotoxins secreted by killer yeasts are capable of killing other strainsof yeast, fungi, or bacteria. For example, microorganisms that can becontrolled by killer yeast include Fusarium and other filamentous fungi.Such yeasts can include, but are not limited to, Wickerhamomyces (e.g.,W. anomalus), Pichia (e.g., P. anomala, P. guielliermondii, P.occidentalis, P. kudriavzevii), Hansenula, Saccharomyces, Hanseniaspora,(e.g., Huvarum), Ustilago (e.g., U. maydis), Debaryomyces hansenii,Candida, Cryptococcus, Kluyveromyces, Torulopsis, Williopsis,Zygosaccharomyces (e.g., Z. bailii), and others.

In one embodiment, the yeast fermentation product can be obtained viacultivation of a biochemical-producing yeast, such as, for example,Pichia anomala (Wickerhamomyces anomalus). Wickerhamomyces anomalus isfrequently associated with food and grain production and is an effectiveproducer of various solvents, enzymes, toxins, as well as glycolipidbiosurfactants, such as SLP. The fermentation broth after 7 days ofcultivation at 25-30° C. can contain the yeast cell suspension and, forexample, 4 g/L or more of glycolipid biosurfactants.

In one embodiment, the yeast fermentation product can also be obtainedvia cultivation of the biosurfactant-producing yeast, Starmerellabombicola. This species is an effective producer of glycolipidbiosurfactants, such as SLP. The fermentation broth after 5 days ofcultivation at 25° C. can contain the yeast cell suspension and, forexample, 150 g/L or more of glycolipid biosurfactants.

In one embodiment, the yeast fermentation product can comprise thefermentation broth, separated from the yeast cells. In one embodiment,the biosurfactants or other growth by-products in the broth are furtherseparated from the broth and purified.

Other microbial strains including, for example, other fungal strainscapable of accumulating significant amounts of, for example, glycolipidor lipopeptide biosurfactants or other metabolites can be used inaccordance with the subject invention. Other metabolites usefulaccording to the present invention include mannoprotein, beta-glucan andothers that have bio-emulsifying and surface/interfacialtension-reducing properties.

Preparation of Microbe-Based Products

One microbe-based product of the subject invention is simply thefermentation broth containing the microorganism and/or the microbialmetabolites produced by the microorganism and/or any residual nutrients.The product of fermentation may be used directly without extraction orpurification. If desired, extraction and purification can be easilyachieved using standard extraction methods or techniques known to thoseskilled in the art.

The microorganisms in the microbe-based product may be in an active orinactive form. The microbe-based products may be used without furtherstabilization, preservation, and storage. Advantageously, direct usageof these microbe-based products preserves a high viability of themicroorganisms, reduces the possibility of contamination from foreignagents and undesirable microorganisms, and maintains the activity of theby-products of microbial growth.

The microbes and/or broth resulting from the microbial growth can beremoved from the growth vessel and transferred via, for example, pipingfor immediate use.

Advantageously, in accordance with the subject invention, themicrobe-based product may comprise broth in which the microbes weregrown. The product may be, for example, at least, by weight, 1%, 5%,10%, 25%, 50%, 75%, or 100% broth. The amount of biomass in the product,by weight, may be, for example, anywhere from 0% to 100% inclusive ofall percentages therebetween.

In other embodiments, the composition (microbes, broth, or microbes andbroth) can be placed in containers of appropriate size, taking intoconsideration, for example, the intended use, the contemplated method ofapplication, the size of the fermentation tank, and any mode oftransportation from microbe growth facility to the location of use.Thus, the containers into which the microbe-based composition is placedmay be, for example, from 1 gallon to 1,000 gallons or more. In certainembodiments the containers are 2 gallons, 5 gallons, 25 gallons, orlarger.

Upon harvesting the microbe-based composition from the growth vessels,further components can be added as the harvested product is placed intocontainers and/or piped (or otherwise transported for use). Theadditives can be, for example, buffers, carriers, other microbe-basedcompositions produced at the same or different facility, viscositymodifiers, preservatives, nutrients for microbe growth, tracking agents,pesticides, and other ingredients specific for an intended use.

Up to, for example, 50 wt. % or more of additives may be added, asneeded, for particular applications, such as, e.g., to vary the VOClevels, increase penetration of the mixture, decrease viscosity of themixture, as couplers for solvent insolubles in the mixture, and toprovide solvents. All additives should have a flash point greater than100° F., preferably greater than 150° F. and more preferably 195° F. TCCto achieve a final product flash point greater than 200° F.

Optionally, the product can be stored prior to use. The storage time ispreferably short. Thus, the storage time may be less than 60 days, 45days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2days, 1 day, or 12 hours. In a preferred embodiment, if live cells arepresent in the product, the product is stored at a cool temperature suchas, for example, less than 20° C., 15° C., 10° C., or 5° C. On the otherhand, a biosurfactant composition can typically be stored at ambienttemperatures.

Local Production of Microbe-Based Products

In preferred embodiments of the subject invention, a microbe growthfacility produces fresh, high-density microorganisms and/or microbialgrowth by-products of interest on a desired scale. The microbe growthfacility may be located at or near the site of application. The facilityproduces high-density microbe-based compositions in batch,quasi-continuous, or continuous cultivation.

In subject invention utilizes cultivation processes ranging from small(e.g., lab setting) to large (e.g., industrial setting) scales. Thesecultivation processes include, but not limited to, submergedcultivation/fermentation, solid state fermentation (SSF), andcombination thereof.

The microbe growth facilities of the subject invention produces fresh,microbe-based compositions, comprising the microbes themselves,microbial metabolites, and/or other components of the broth in which themicrobes are grown. If desired, the compositions can have a high densityof vegetative cells or a mixture of vegetative cells, spores, mycelia,conidia, or other microbial propagules.

Advantageously, the subject microbe-based products can be tailored foruse at a specified location. In one embodiment, the microbe growthfacility is located on, or near, a site where the microbe-based productswill be used. For example, the microbe growth facility may be less than300, 250, 200, 150, 100, 75, 50, 25, 15, 10, 5, 3, or 1 mile from thelocation of use.

Because the microbe-based product is generated locally, on-site or nearthe site of application, without resort to the microorganismstabilization, preservation, storage and transportation processes ofconventional microbial production, a much higher density of livemicroorganisms can be generated. Thus, a smaller volume of themicrobe-based product is required for use in the on-site application.Furthermore, this allows for higher density microbial applications wherenecessary to achieve the desired efficacy.

Advantageously, this allows for a scaled-down bioreactor (e.g., smallerfermentation tank, and smaller supplies of starter material, nutrients,pH control agents, and de-foaming agents, etc.), which makes the systemefficient and facilitates the portability of the product. Localgeneration of the microbe-based product also facilitates the inclusionof the growth broth in the product, thus eliminating the requirement forstabilizing cells or separating them from their culture broth. The brothcan contain agents produced during the fermentation that areparticularly well-suited for local use.

Locally-produced high density, robust cultures of microbes are moreeffective in the field than those that have undergone cell stabilizationor have been sitting in the supply chain for some time. Themicrobe-based products of the subject invention are particularlyadvantageous compared to traditional products wherein cells, spores,mycelia, conidia or other microbial propagules have been separated frommetabolites and nutrients present in the fermentation growth media.Reduced transportation times allow for the production and delivery offresh batches of microbes and/or their metabolites at the time andvolume as required by local demand.

Advantageously, these microbe growth facilities provide a solution tothe current problem of relying on far-flung industrial-sized producerswhose product quality suffers due to upstream processing delays, supplychain bottlenecks, improper storage, and other contingencies thatinhibit the timely delivery and application of, for example, a viable,high cell-count product and the associated broth and metabolites inwhich the cells are originally grown.

The microbe growth facilities provide manufacturing versatility by theability to tailor the microbe-based products to improve synergies withdestination geographies. Advantageously, in preferred embodiments, thesystems of the subject invention harness the power ofnaturally-occurring local microorganisms and their metabolic by-productsto improve oil production. Local microbes can be identified based on,for example, salt tolerance, or ability to grow at high temperatures.

The cultivation time for the individual vessels may be, for example,from 1 to 7 days or longer. The cultivation product can be harvested inany of a number of different ways.

Local production and delivery within, for example, 24 hours offermentation results in pure, high cell density compositions andsubstantially lower shipping costs. Given the prospects for rapidadvancement in the development of more effective and powerful microbialinoculants, consumers will benefit greatly from this ability to rapidlydeliver microbe-based products.

Methods of Enhanced Oil Recovery

In some embodiments, the subject invention provides materials andmethods for improving oil production by treating an oil-producing site,e.g., an oil-bearing formation or an oil well, with microorganismsand/or their growth by-products. In one embodiment, the subjectinvention can be useful for enhancing oil recovery from an oil well by,e.g., stimulating the flow of oil from the well while dissolving scalewithin the formation.

As used herein, “applying” a composition or product refers to contactingit with a target or site such that the composition or product can havean effect on that target or site. The effect can be due to, for example,microbial growth and/or the action of a biosurfactant or other growthby-product. For example, the microbe-based compositions or products canbe injected into oil wells and/or the piping, casing, annulus, pumps,tanks, etc. associated with oil-producing sites and oil-bearingformations.

In one embodiment the subject invention provides a method for improvingoil recovery by applying one or more microorganisms capable of producinguseful biochemical byproducts to an oil producing site, e.g., anoil-bearing formation and/or oil well. The method optionally includesadding nutrients and/or other agents to the site. In preferredembodiments, the microorganism is a biosurfactant-producing species ofbacteria.

In certain embodiments, the microorganisms are selected from strains ofBacillus, including, but not limited to, strains of Bacillus subtilis,Bacillus licheniformis, and Bacillus amyloliquefaciens. In preferredembodiments, the bacteria are in spore form.

In one embodiment, the method further comprises adding nutrients and/orgermination enhancers to promote microbial germination and growth. Forexample, nutrients such as sources of carbon, nitrogen, magnesium,phosphorous and protein can be added. Germination enhancers, such asL-alanine and manganese, can also be added.

The method may also comprise adding a yeast fermentation product, suchas the fermentation broth resulting from cultivation of, e.g.,Starmerella bombicola or Wickerhamomyces anomalus. In one embodiment,the yeast is a biosurfactant-producing yeast. In one embodiment, thefermentation broth comprises the by-products of yeast growth, such as,for example, glycolipid biosurfactants and other metabolites.

In one embodiment, the yeast cells can be removed from the yeastfermentation product and only the broth containing biosurfactants andother metabolites is applied. In one embodiment, the yeast fermentationproduct comprises biosurfactants that have been separated from thefermentation broth and purified.

In certain embodiments, the yeast fermentation products of the subjectinvention have advantages over, for example, biosurfactants alone,including one or more of the following: high concentrations ofmannoprotein as a part of a yeast cell wall's outer surface; thepresence of beta-glucan in yeast cell walls; and the presence ofbiosurfactants and other metabolites (e.g., lactic acid, ethanol, ethylacetate, etc.) in the culture.

The method may also comprise applying the microbes and/or microbialgrowth by-products with one or more alkaline compounds. The alkalinecompound can be, for example, ammonium hydroxide.

In some embodiments, the method may also comprise applying the microbesand/or microbial growth by-products with one or more polymer compounds.The polymer compounds can be selected from biopolymers such as, forexample, hydrogels, polysaccharides, xanthan gum, guar gum, andcellulose polymers.

In some embodiments, the method may also comprise applying the microbesand/or microbial growth by-products with one or more non-biologicalsurfactants. The surfactants may be, for example, anionic, cationic,non-ionic or zwitterionic.

In one embodiment, the microbes and/or microbial growth by-products canbe applied with one or more chelating agents for reducing, e.g.,dissolving, scale that has accumulated within the oil-bearing formation.The chelating agents may be, for example, citric acid, EDTA, and/orsodium citrate.

In some embodiments, the subject microbe-based products and methods canfurther be used for paraffin removal, liquefaction of solid asphaltene,and bioremediation of hydrocarbon-contaminated waters, soils and othersites.

In one embodiment, the microbe-based products are applied to a workingwell, including the surrounding formation. In this embodiment, theproduct can be poured or injected down the casing side (back lines) of awell and allowing it to mix with the fluid that is already in the well.When enough fluid is present, the composition can then optionally becirculated by, for example, a pump for 24-72 hours, preferably 48-72hours. Prior to circulating, the composition may be allowed to set for 8to 24 hours, for example. The setting time, circulating time and dosagedepend on the depth and size of the well. A basic initial dosage can be,but is not limited to, 20 gallons of composition, and at least about 5gallons of composition per well on periodic basis, e.g. biweekly,monthly, bimonthly.

In one embodiment, the microorganisms can germinate and grow in situ andproduce biosurfactants in the oil-producing site. Consequently, a highconcentration of biosurfactants and biosurfactant-producingmicroorganisms at a treatment site (e.g., an oil well) can be achievedeasily and continuously.

In one embodiment, it is desirable to introduce the composition, throughperforations in the casing, into the surrounding oil-bearing formation.The composition may be forced into the surrounding formation by appliedpressure or, if the composition is allowed to set at the bottom of thecasing, the composition may seep into the formation without additionalpressure. The composition permeates the formation, dissolving blockagesin the formation to provide more efficient oil and gas recovery.

In additional embodiments, the composition of the subject invention maybe applied directly to equipment. For example, prior to placing rods andcasings into gas and/or oil wells, these parts may be sprayed with, orsoaked in, the composition. The parts may also be dipped into tanksfilled with the composition.

The composition may be introduced by means of injection pumps intooff-shore gas or oil wells to enhance oil recovery. To treat the lines,from 1-500 gallons up to 1000 barrels, 10,000 barrels, or more, forexample, of the composition can be applied to the composition at aninjection rate of, for example, 1 to 20 gallons per minute, or 1 to 20barrels per minute.

The subject treatment can be effective in a range of different geologicformations. For example, the subject invention can be useful informations as deep as about 7,000 feet or deeper, and as shallow asabout 1,500 feet or shallower. Additionally, the invention can be usefulin formations having a range of porosity and/or permeability, forexample from about 0.1% to about 20% or more. The invention can also beuseful in formations having a wide range of temperatures, pH, andsalinity.

In one embodiment, enhanced oil recovery is achieved through selectiveplugging, wherein fluid flow through the reservoir is shifted from thereservoir's high permeability zones to moderate or low permeabilityzones. Sweep efficiency can be increased by, for example, forcinginjected water to pass through previously by-passed oil zones of thereservoir. The changes in flow pattern can be achieved by an increase inmicrobial cell mass within the reservoir by, for example, injectingmicroorganisms together with nutrients. The injected nutrient andmicrobes preferentially flow into the high permeability zones of thereservoir and as a result of cell growth, the biomass selectively plugsthese zones to a greater extent than the moderate or low permeabilityzones. In one embodiment, the microbes are injected in spore form andgerminate while inside the reservoir.

Enhanced Oil Recovery Via the Alkaline-Surfactant-Polymer (ASP) Method

In one embodiment, methods for enhancing oil recovery are provided,wherein a microbe-based product of the subject invention is applied toan oil production site in combination with one or more alkalinecompounds, polymers, surfactants, or combinations thereof.

In surfactant flooding, by reducing the interfacial tension between theoil and the displacing water and also the interfacial tension betweenthe oil and the rock interfaces, residual oil can be displaced andrecovered.

In caustic flooding, the reaction of the alkaline compounds with theorganic acids in the oil forms in situ natural surfactants that lowerthe oil-water interfacial tension.

In addition to surfactant and alkaline flooding, polymers are used toincrease the viscosity of the displacing water to improve the oil sweptefficiency.

ASP flooding is a combination process in which alkali, surfactant andpolymer are injected. ASP involves the injection of a solutioncontaining polymer, alkali and surfactant into a depleted or maturedoilfield with the objective of achieving optimum chemistry at largeinjection volumes for minimum cost. The alkali-surfactant mixture formsan emulsion with the oil, which is then swept and displaced from thereservoir using a polymer drive. ASP flooding improves microscopicdisplacement efficiency by reducing the interfacial tension (IFT)between the water and oil through the addition of a surfactant to thewater, while matching the oil and water mobility through the addition ofpolymer. Alkali is also added to the water to reduce adsorption of thesurfactant onto the pore walls and to control the local salinity toensure minimum IFT and alter the rock wettability.

Use of Microbes with Surfactants in Oil Recovery

In certain embodiments, the methods of recovering oil described hereinutilize one or more microbes and/or microbial growth by-products (e.g.,biosurfactants), combined with other compositions. In one embodiment,the other compositions are non-biological surfactants.

A surfactant (Surface Active Agent) molecule has two functional groups,namely a hydrophilic (water-soluble) or polar group and a hydrophobic(oil-soluble) or non-polar group. The hydrophobic group is usually along hydrocarbon chain (C8-C18), which may or may not be branched, whilethe hydrophilic group is formed by moieties such as carboxylates,sulfates, sulfonates (anionic), alcohols, polyoxyethylenated chains(nonionic) and quaternary ammonium salts (cationic).

Surfactants work in ASP flooding to lower the interfacial tension (IFT)between trapped oil and brine, to aid mobilization and contribute to theformation of oil banks. IFT reduction lowers capillary forces and allowsfor the oil bank to flow more freely without renewed trapping. Theselection of an appropriate surfactant for EOR purposes is based on theability to reduce IFT between crude and brine, thermal stability,tolerance to salinity and hardness of brine, solubility in brine, phasebehavior parameters, adsorption test under static and dynamic conditionand displacement studies under reservoir conditions.

Surfactants according to the subject methods include, but are notlimited to: anionic surfactants, ammonium lauryl sulfate, sodium laurylsulfate (also called SDS, sodium dodecyl sulfate), alkyl-ether sulfatessodium laureth sulfate (also known as sodium lauryl ether sulfate(SLES)), sodium myreth sulfate; docusates, dioctyl sodiumsulfosuccinate, perfluorooctanesulfonate (PFOS),perfluorobutanesulfonate, linear alkylbenzene sulfonates (LABs),alkyl-aryl ether phosphates, alkyl ether phosphate; carboxylates, alkylcarboxylates (soaps), sodium stearate, sodium lauroyl sarcosinate,carboxylate-based fluorosurfactants, perfluorononanoate,perfluorooctanoate; cationic surfactants, pH-dependent primary,secondary, or tertiary amines, octenidine dihydrochloride, permanentlycharged quaternary ammonium cations, alkyltrimethylammonium salts, cetyltrimethylammonium bromide (CTAB) (a.k.a. hexadecyl trimethyl ammoniumbromide), cetyl trimethylammonium chloride (CTAC), cetylpyridiniumchloride (CPC), benzalkonium chloride (BAC), benzethonium chloride(BZT), 5-Bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammoniumchloride, cetrimonium bromide, dioctadecyldi-methylammonium bromide(DODAB); zwitterionic (amphoteric) surfactants, sultaines CHAPS(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate),cocamidopropyl hydroxysultaine, betaines, cocamidopropyl betaine,phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine,sphingomyelins, ethoxylate, long chain alcohols, fatty alcohols, cetylalcohol, stearyl alcohol, cetostearyl alcohol, oleyl alcohol,polyoxyethylene glycol alkyl ethers (Brij):CH3-(CH2)10-16-O—C2H4)1-25-OH (octaethylene glycol monododecyl ether,pentaethylene glycol monododecyl ether), polyoxypropylene glycol alkylethers: CH3-(CH2)10-16-(O—C3H6)1-25-OH, glucoside alkyl ethers:CH3-(CH2)10-16-(O-Glucoside)1-3-OH (decyl glucoside, lauryl glucoside,octyl glucoside), polyoxyethylene glycol octylphenol ethers:C8H17-(C6H4)-(O—C2H4)1-25-OH (Triton X-100), polyoxyethylene glycolalkylphenol ethers: C9H19-(C6H4)-(O—C2H4)1-25-OH (nonoxynol-9), glycerolalkyl esters (glyceryl laurate), polyoxyethylene glycol sorbitan alkylesters (polysorbate), sorbitan alkyl esters (spans), cocamide MEA,cocamide DEA, dodecyldimethylamine oxide, copolymers of polyethyleneglycol and polypropylene glycol (poloxamers), and polyethoxylated tallowamine (POEA).

Anionic surfactants contain anionic functional groups at their head,such as sulfate, sulfonate, phosphate, and carboxylates. Prominent alkylsulfates include ammonium lauryl sulfate, sodium lauryl sulfate (alsocalled SDS, sodium dodecyl sulfate) and the related alkyl-ether sulfatessodium laureth sulfate, also known as sodium lauryl ether sulfate(SLES), and sodium myreth sulfate. Carboxylates are the most commonsurfactants and comprise the alkyl carboxylates (soaps), such as sodiumstearate.

Surfactants with cationic head groups include: pH-dependent primary,secondary, or tertiary amines; octenidine dihydrochloride; permanentlycharged quaternary ammonium cations such as alkyltrimethylammoniumsalts: cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethylammonium bromide, cetyl trimethylammonium chloride (CTAC);cetylpyridinium chloride (CPC); benzalkonium chloride (BAC);benzethonium chloride (BZT); 5-Bromo-5-nitro-1,3-dioxane;dimethyldioctadecylammonium chloride; cetrimonium bromide; anddioctadecyldi-methylammonium bromide (DODAB).

Zwitterionic (amphoteric) surfactants have both cationic and anioniccenters attached to the same molecule. The cationic part is based onprimary, secondary, or tertiary amines or quaternary ammonium cations.The anionic part can be more variable and include sulfonates. The mostcommon biological zwitterionic surfactants have a phosphate anion withan amine or ammonium, such as the phospholipids phosphatidylserine,phosphatidylethanolamine, phosphatidylcholine, and sphingomyelins.

A surfactant with a non-charged hydrophilic part, e.g. ethoxylate, isnon-ionic. Many long chain alcohols exhibit some surfactant properties.

Use of Microbes with Polymers in Oil Recovery

The present invention provides for methods of enhanced oil recoveryusing one or more microbes and/or microbial growth by-products, combinedwith one or more polymer compounds. Polymer compounds used to recoveroil in combination with the microbes of the present invention includebut are not limited to: hydrogels, acrylic acid, acrylamide,polyacrylamide (PAM), hydrolyzed polyacrylamide (HPAM), polysaccharide,xanthan gum, guar gum, and cellulose polymer. In preferred embodiments,the polymer is a biopolymer selected from, for example, hydrogels,xanthan gum, guar gum, cellulose polymers, polysaccharides, and others.

The associative water-soluble polymer is a relatively new class ofpolymers that has recently been introduced for oilfield applications.These polymers consist of a hydrophilic long-chain backbone, with asmall number of hydrophobic groups localized either randomly along thechain or at the chain ends. When these polymers are dissolved in water,hydrophobic groups aggregate to minimize their water exposure. Theincorporated groups associate due the intramolecular hydrophobicinteractions and the intermolecular hydrophobic interactions. Thefunctional groups on these polymer are less sensitive to brine salinitycompared to polyacrylamide, whose viscosity dramatically decreases withincreasing salinity.

Polymer flooding may involve addition of polymer to the water of awater-flood to decrease its mobility. Polymers increase the viscosity ofthe aqueous phase as well as reduces water permeability due tomechanical entrapment, consequently resulting in more favorable mobilityratio. With a more viscous phase, the collected oil bank can be moreeasily moved through the reservoir and eventually into the producingwell.

The polymers according to these embodiments can also be removed and/ordegraded using the microbe-based composition of the subject inventiononce their function in the well is no longer needed.

In one embodiment, the subject invention provides a method for improvinghydrocarbon recovery from a fracking well by applying to a drilling sitethe microbe-based composition comprising one or more strains ofmicroorganisms. In certain embodiments, the polymers have built upinside the well after they have performed their desired function insidethe well.

The microbes of the microbe-based composition and/or their growthbyproducts can quickly digest polymers such as polylactic acid (PLA);thus, the method improves the ability to recover hydrocarbon resourcesby reducing the buildup of PLA and other resins within the fractures andwellbores of fracking wells once their utility has been exhausted. Themethod optionally includes adding nutrients and/or other agents to thesite in order to promote microbial growth. The method further caninclude adding polymer-degrading enzymes to the site in order to enhancepolymer degradation.

In one embodiment, the subject invention provides methods of recoveringpolymeric substances that remain in wells, including fracking wells,after their utility has been exhausted. For example, biosurfactantsproduced by methods and microorganisms of the present invention canreduce interfacial tension of fluids used for uplifting polymericsubstances, such as PAM gel friction reducers. In another embodiment,the biosurfactants can be used to cleave PAM gel prior to uplifting.

Use of Microbes with Alkaline Compounds in Oil Recovery

The present invention provides for methods of enhanced oil recoveryusing one or more microbes and/or microbial growth by-products, combinedwith one or more alkaline compounds. Alkaline compounds used to recoveroil in combination with the microbes of the present invention includebut are not limited to ammonium hydroxide.

Alkali is a basic, ionic salt of an alkali metal or alkaline earth metalelement. The use of alkali in a chemical flood offers several benefitsincluding promoting crude oil emulsification, increasing aqueous-phaseionic strength leading to regulation of phase behavior of the injectedsurfactant, and lowering IFT to ultralow values in presence ofsurfactant.

Alkali can also reduce costs by limiting the amount of surfactant neededin two ways. First, alkali reduces surfactant adsorption by increasingthe rock surface's negative charge density, making it preferentiallywater-wet. Second, alkali reacts with the acids in the crude oils toproduce in situ soaps, which in turn broadens the optimal salinityrange. The soap generated creates a microemulsion phase that canco-exist with oil and water, thus extending the three-phase region (orultra-low IFT region).

Selection of alkali is guided by the type of formation, clay type anddivalent cations. Common alkaline agents include sodium hydroxide (NaOH,or caustic soda), sodium carbonate (Na₂CO₃, or soda ash), sodiumbicarbonate (NaHCO₃) and sodium metaborate (NaBO₂). Sodium hydroxidesolutions have been reported to strongly interact with sandstone atelevated temperature (185° F.), resulting in sandstone weight loss andincreased porosity. Caustic consumption resulting from NaOH dissolutionof silicate minerals can be a significant and detrimental factor duringfield application. Anionic surfactants showed much smaller adsorption inthe presence of Na₂CO₃ compared to NaOH. The hydroxide is not apotential determining ion for carbonate surfaces. Calcium and otherdivalent cations can cause precipitation of alkalis such as Na₂CO₃unless soft brine is used. This is limitation of Na₂CO₃. The use ofNaBO₂ as a replacement for Na₂CO₃ has been reported. This alkali gave pHvalues of about 11 at 1 wt % alkali concentration and generated soap foracidic crude oils. Another major advantage of NaBO₂ (sodium metaborate)species are their tolerance to divalent cations. In carbonate reservoirssodium metaborate is used in place of other alkalis. If reservoircontains clays NaHCO₃ is preferred. Na₂CO₃ is the most commonly usedalkali because it is inexpensive and transports better in porous media.

The preferred oil formations for alkaline flooding are sandstonereservoirs rather than carbonate formations that contain anhydrite(calcium sulfate) (CaSO₄) or gypsum (calcium sulfate dehydrate)(CaSO₄.2H₂O), which can consume large amounts of alkaline chemicals.Also, in carbonate reservoirs the calcium carbonate (CaCO₃) or calciumhydroxide (Ca(OH)₂) precipitation occurs when Na₂CO₃ or NaOH is added.Carbonate reservoirs also contain brine with a higher concentration ofdivalents and could cause precipitation. To overcome this problem,suggested NaHCO₃ and sodium sulfate (Na₂SO₄) is used. NaHCO₃ has a muchlower carbonate ion concentration, and additional sulfate ions candecrease calcium ion concentration in the solution. These chemicals arealso consumed by clays, minerals, or silica, and the higher thetemperature of the reservoir the higher the alkali consumption. Anothercommon problem during caustic flooding is scale formation in theproducing wells. During alkaline flooding, the injection sequenceusually includes: (1) a preflush to condition the reservoir beforeinjection of the primary slug, (2) primary slug (alkaline chemicals),(3) polymer as a mobility buffer to displace the primary slug. Alkalineflooding can be modified as the AP (alkali-polymer), AS(alkali-surfactant), and Alkali-Surfactant-Polymer (ASP) processes. Soapproduced from the reaction between the acidic components of a crude oiland the injected alkali is the principal mechanism of oil recovery inalkaline flooding.

Use of Microbes with Chelating Agents

In some embodiments, the microorganisms and/or growth by-productsthereof can be applied with a chelator or chelating agent.Advantageously, the use of chelating agents aides in enhancing oilrecovery by dissolving scale within a formation. Scale can block thepores and other flow paths of an oil-bearing formation, thus slowingand/or blocking the flow of oil from the formation.

As used herein, “chelator” or “chelating agent” means an active agentcapable of removing a metal ion from a system by forming a complex sothat the metal ion, for example, cannot readily participate in orcatalyze oxygen radical formation.

Examples of chelating agents suitable for the present invention include,but are not limited to, dimercaptosuccinic acid (DMSA),2,3-dimercaptopropanesulfonic acid (DMPS), alpha lipoic acid (ALA),thiamine tetrahydrofurfuryl disulfide (TTFD), penicillamine,ethylenediaminetetraacetic acid (EDTA), sodium acetate, sodium citrateand citric acid.

In preferred embodiments, the chelating agent is sodium citrate, citricacid, EDTA or a combination thereof.

EXAMPLES

A greater understanding of the present invention and of its manyadvantages may be had from the following examples, given by way ofillustration. The following examples are illustrative of some of themethods, applications, embodiments and variants of the presentinvention. They are not to be considered as limiting the invention.Numerous changes and modifications can be made with respect to theinvention.

Example 1 Production of Bacillus Subtilis

Fermentation of Bacillus subtilis var. lotuses can be performed in a 500L reactor with 350 L of a nutrient medium containing (g/L):

Glucose 18 Powder molasses 2 Sucrose 1 KH₂PO₄ 0.5 Na₂HPO₄•7H₂O 2.1 KCl0.1 MgSO₄ 0.5 CaCl₂ 0.05 Urea 2.5 NH₄Cl 1.24 Yeast extract 2 Cornpeptone 0.5 TekNova trace element (mL) 1

Temperature of cultivation is 40° C., pH stabilization is from 6.8-7.0,and DO stabilization is at 20-30% (concentration of oxygen in the air istaken as 100%). Duration of cultivation is 24-36 hours, or until atleast 95% of the bacteria reach sporulation. The final concentration ofbacterial culture is no less than 1×10⁹ CFU/ml. The amount of culturemanufactured by a single fermentation cycle allows for the production ofmore than 2,000 barrels of final treatment formulation containing 10⁶CFU of this strain of Bacillus.

Example 2 Fermentation of Starmerella Bombicola for BiosurfactantProduction

The fermenter is an autoclavable stainless steel vessel designedspecifically for cultivation of yeast and production of biosurfactants.The fermenter is fitted with a microsparger and an impeller, as well aswith dissolved oxygen, pH, temperature and foam probes. It has anintegrated control station with a color touchscreen interface, built-inpumps for enhanced mixture of broth, gas flow controllers, and pH/DOfoam/level controllers. The working volume of the 550 gallon reactor is500 gallons.

The nutrient medium contains sources of carbon, protein, nitrogen, andunsaturated oil or fatty acids. One-day old culture of Starmerellabombicola (60-70 L) is used to inoculate the reactor. Initial pH of theculture is 5.0-6.0 until growth of microbes occurs and pH begins todecline. Cultivation duration and readymade product collection continuefor 5 days at 25° C. and pH is stabilized at 3.5. The final sophorolipidcontent can reach at least 40% of working volume per cycle, or 150 g/Lor higher.

What is claimed:
 1. A method for enhancing the amount of oil recoverablefrom an oil-bearing formation, wherein the method comprises: applying tothe formation a yeast fermentation product comprising a broth in which aWickerhamomyces anomalus or Starmerella bombicola yeast was cultivated,wherein said broth comprises a glycolipid biosurfactant produced by saidyeast, and wherein said broth contains yeast cellular components but noliving yeasts; wherein said method further comprises applying to theformation: a solvent selected from the group consisting of isopropylalcohol and ethanol; and a chelating agent comprising a mixture of twoor more of citric acid, sodium citrate, and EDTA.
 2. The method of claim1, further comprising administering one or more polymer compounds to theformation.
 3. The method of claim 2, wherein the one or more polymercompounds are selected from the group consisting of xanthan gum, guargum, hydrogels, polysaccharide, and cellulose polymers.
 4. The method ofclaim 1, further comprising administering one or more non-biologicalsurfactants to the formation.
 5. The method of claim 1, wherein themethod enhances oil recovery and also dissolves scale deposits presentin the formation.
 6. The method of claim 1, wherein the yeastfermentation product, the solvent and the chelating agent are mixed withwater prior to application, and wherein the glycolipid biosurfactant isa sophorolipid and the solvent is isopropyl alcohol.
 7. The method ofclaim 1, wherein the yeast is Starmerella bombicola.